Targeting specificity of nuclear-encoded organelle proteins with a self-assembling split-fluorescent protein toolkit

Abstract

A large number of nuclear-encoded proteins are targeted to the organelles of endosymbiotic origin, namely mitochondria and plastids. To determine the targeting specificity of these proteins, fluorescent protein tagging is a popular approach. However, ectopic expression of fluorescent protein fusions commonly results in considerable background signals and often suffers from the large size and robust folding of the reporter protein, which may perturb membrane transport. Among the alternative approaches that have been developed in recent years, the self-assembling split-fluorescent protein (sasplit-FP) technology appears particularly promising to analyze protein targeting specificity in vivo. Here, we improved the sensitivity of this technology and systematically evaluated its utilization to determine protein targeting to plastids and mitochondria. Furthermore, to facilitate high-throughput screening of candidate proteins we developed a Golden Gate-based vector toolkit (PlaMinGo). As a result of these improvements, dual targeting could be detected for a number of proteins that had earlier been characterized as being targeted to a single organelle only. These results were independently confirmed with a plant phenotype complementation approach based on the immutans mutant. This article has an associated First Person interview with the first author of the paper.

Dual targeting of TatA points to a chloroplast-like Tat pathway in plant mitochondria

“…The two non-fluorescent fragments can assemble spontaneously to reconstitute the functional fluorophore. If combined with specific organelle-targeting signals, the self-assembly property can be used for subcellular localization studies (Sharma et al 2019; see also suppl. Fig.…”

“…The subcellular localization of TatA was analyzed by combining the N-terminal 100 residues of peaTatA with a sevenfold repeat of GFP11 (GFP11 x7 ) (Sharma et al, 2019). The resulting chimera (peaTatA 1-100 /GFP11 x7 ) was co-infiltrated with the organelle-specific constructs FNR 1-55 /GFP1-10 or mtRi 1-100 /GFP1-10, which mediate transport of the “receptor” component specifically into chloroplasts and mitochondria, respectively.…”

“…While GFP reconstitution within chloroplasts was always observed in a large number of cells after transformation, fluorescence signals in mitochondria could be detected with only low frequency in all repetitions. This is not a matter of lacking reconstitution in mitochondria in general because GFP fluorescence was observed at high frequency with the mtRi 1-100 /GFP11 x7 construct (Sharma et al 2019; see also suppl. Fig.…”

“…Likewise, the generation of the sa split/GFP reporter constructs, namely FNR 1-55 /GFP1-10, FNR 1-55 /GFP11 x7 , mtRi 1-100 /GFP1-10, and mtRi 1-100 /GFP11 x7 , are described in Sharma et al (2019). The coding sequence of eYFP in pRT100peaTatA 1-100 /eYFP was replaced by GFP11 x7 via restriction-free cloning (Bond and Naus, 2012) generating pRT100peaTatA 1-100 /GFP11 x7 .…”

“…Particle bombardment of 7 - 10 days old pea seedlings was carried out according to Sharma et al (2018). Agrobacterium infiltration of eYFP reporter constructs and sasplitGFP vectors into the lower epidermis of Nicotiana benthamiana leaves were performed as described (Sharma et al, 2018 and 2019, respectively). The mitochondria of epidermal cells were stained by infiltrating MitoTracker Orange (0.1 μM MitoTracker Orange® CMTMRos in 10 mM MgCl 2 , 10 mM MES, pH 5.6) into the lower epidermis of leaf tissue and imaged 15 min after infiltration.…”